Effects of dietary marjoram, Origanum majorana extract on growth performance, hematological, antioxidant, humoral and mucosal immune responses, and resistance of common carp, Cyprinus carpio against Aeromonas hydrophila

Journal Pre-proof Effects of dietary marjoram, Origanum majorana extract on growth performance, hematological, antioxidant, humoral and mucosal immune responses, and resistance of common carp, Cyprinus carpio against Aeromonas hydrophila Morteza Yousefi, Hamed Ghafarifarsani, Seyed Hossein Hoseinifar, Ghasem Rashidian, Hien Van Doan PII:

S1050-4648(20)30724-5

DOI:

https://doi.org/10.1016/j.fsi.2020.11.019

Reference:

YFSIM 7365

To appear in:

Fish and Shellfish Immunology

Received Date: 20 August 2020 Revised Date:

13 November 2020

Accepted Date: 18 November 2020

Please cite this article as: Yousefi M, Ghafarifarsani H, Hoseinifar SH, Rashidian G, Van Doan H, Effects of dietary marjoram, Origanum majorana extract on growth performance, hematological, antioxidant, humoral and mucosal immune responses, and resistance of common carp, Cyprinus carpio against Aeromonas hydrophila, Fish and Shellfish Immunology (2020), doi: https://doi.org/10.1016/ j.fsi.2020.11.019. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

Seyed Hossein Hoseinifar conceived and designed the experiments. Hamed Ghafarifarsani and Ghasem Rashidian performed the experiments. Hien Van Doan analyzed the data. Morteza Yousefi wrote and revised the paper. All authors read

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and approved the final manuscript.

Effects of dietary marjoram, Origanum majorana extract on growth performance, hematological, antioxidant, humoral and mucosal immune responses, and resistance of common carp, Cyprinus carpio against Aeromonas hydrophila Running head: Dietary marjoram effects on common carp

Morteza Yousefi a, Hamed Ghafarifarsani b, Seyed Hossein Hoseinifar c, Ghasem Rashidian d, Hien

Department of Veterinary Medicine, Peoples' Friendship University of Russia (RUDN University), 6

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Van Doan e,f*

Department of Fisheries, Faculty of Agriculture and Natural Resources, Urmia University, Urmia,

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Miklukho-Maklaya St, Moscow 117198, Russian Federation.

Department of Fisheries, Faculty of Fisheries and Environmental Sciences, Gorgan University of

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Iran.

Agricultural Sciences and Natural Resources, Gorgan, Iran. Aquaculture Department, Faculty of Marine Sciences, Tarbiat Modares University, Noor, Iran.

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Department of Animal and Aquatic Sciences, Faculty of Agriculture, Chiang Mai University, Chiang

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Mai 50200 Thailand. f

Science and Technology Research Institute, Chiang Mai University 239 Huay Keaw Rd., Suthep,

Muang, Chiang Mai 50200, Thailand. * Corresponding author: * Tel.: +66 90-029-9995; [email protected]

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Effects of dietary marjoram, Origanum majorana extract on growth performance, hematological,

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antioxidant, humoral and mucosal immune responses, and resistance of common carp, Cyprinus

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carpio against Aeromonas hydrophila

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Running head: Dietary marjoram effects on common carp

5 Morteza Yousefi a, Hamed Ghafarifarsani b, Seyed Hossein Hoseinifar c, Ghasem Rashidian d,

7

Hien Van Doan e,f*

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*a

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University), 6 Miklukho-Maklaya St, Moscow 117198, Russian Federation.

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Department of Veterinary Medicine, Peoples' Friendship University of Russia (RUDN

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b

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Urmia, Iran.

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c

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of Agricultural Sciences and Natural Resources, Gorgan, Iran.

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Aquaculture Department, Faculty of Marine Sciences, Tarbiat Modares University, Noor, Iran.

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Department of Animal and Aquatic Sciences, Faculty of Agriculture, Chiang Mai University,

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Chiang Mai 50200 Thailand.

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Suthep, Muang, Chiang Mai 50200, Thailand.

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* Corresponding author:

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* Tel.: +66 90-029-9995; [email protected]

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Department of Fisheries, Faculty of Agriculture and Natural Resources, Urmia University,

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Department of Fisheries, Faculty of Fisheries and Environmental Sciences, Gorgan University

Science and Technology Research Institute, Chiang Mai University 239 Huay Keaw Rd.,

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Abstract

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The present study aimed to investigate the potential effects of dietary marjoram extract on

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growth performance, health, and disease resistance in common carp, Cyprinus carpio. To this

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purpose, the fish were assigned into four treatments and fed by diets supplemented with 0

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(control), 100, 200, and 400 mg marjoram extract kg-1 over eight weeks and then challenged with

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Aeromonas hydrophila. According to the results, 200 mg kg-1 dietary marjoram extract inclusion

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showed the highest final weight, weight gain (%) and specific growth rate, and lowest feed

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conversion ratio (FCR). White blood cell number, Red blood cells, hematocrit, hemoglobin,

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mean corpuscular volume and mean corpuscular hemoglobin were markedly increased

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particularly at 200 mg kg-1 treatment. Marjoram extract significantly increased plasma

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superoxide dismutase activity and decreased malondialdehyde level compared to the control

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treatment. Plasma complement and lysozyme activities and total immunoglobulin levels,

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mucosal complement, lysozyme and alkaline phosphatase activities and immunoglobulin levels

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were significantly increased compared to the control group. The lowest post-challenge survival

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rate was observed in the control treatment, whereas the highest value was related to the 200 mg

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kg-1 marjoram treatment. In conclusion, the present study demonstrated that, marjoram extract is

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a suitable feed supplements for common carp, as it stimulates the fish growth, antioxidant, and

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immune systems, which suppress the fish mortality during Aeromonas septicemia. According to

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the results, 200 mg marjoram extract kg-1 is recommended for carp feed formulation.

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Keywords: Plant extract, Growth promoter, Immune response, Mucosal immunity, Common

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carp, Fish disease

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1. Introduction

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Aquaculture importantly participates in global protein supply and suppresses the catch pressure

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on natural resources of aquatic organisms. Sustainable aquaculture activity directly correlates to

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fish farm profitability, which could be augmented by increasing growth rate and preventing

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diseases losses [1].

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Aeromonas hydrophila, as a gram-negative bacterium, is an opportunistic fish pathogen that

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infects many species [2–4]. The outbreak of the disease is associated with stressful conditions

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and deteriorated fish health [5]. Antibiotics are used to cure it, but there are several reports about

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the rise of antibiotic-resistant isolates [6–7], the accumulation of residual antibiotics in

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aquaculture products [8], environmental pollution and detrimental effect on the microbial

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biodiversity [9]. Therefore, due to the complicated problems that the overuse of antibiotics has

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created, since the beginning of this century, strictly regulates have been enacted by some

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countries and international institutions to restrict the use of antibiotics. For example, according to

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the United States Food and Drug Administration and European Agency for the Evaluation of

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Medicinal Products, the use of several antibiotics has been prohibited in food-producing animals

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and animal-feed products [10,11]. Therefore, the best choice to counteract the disease is

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managerial issues, which suppress stress and increase health and immune of fish, enabling them

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to resist against A. hydrophila. One of the practical methods to achieve these goals is use of

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dietary supplements with growth-promoting, stress-mitigating, immunostimulating, and

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antioxidant properties [12,13]. Among the various feed supplements, herbal agents have recently

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gained a great attention due to the presence of various bioactive compounds [14–16]. For

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example, dietary supplementation with Olea europea [17], Quercus castaneifolia [18],

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Lavandula angustifolia [19], Camellia sinensis [20], and Ocimum sanctum [21] leaf extract have

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remarkably improved fish growth performance, immune and antioxidant functions, and/or

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resistance against A. hydrophila in different fish species.

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The family Labiatae includes several plants such as Thymus vulgaris and Origanum vulgare,

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well-known plants with growth-promoting, antioxidant and immunostimulating effects in fish

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[22–31]. However, little is known about the potentials of marjoram, Origanum majorana,

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another Labiatae plants, in aquaculture. The plant extract was found to prevent growth of

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different fungi and bacteria [32], including antibiotic-resistant strain of a fish pathogen,

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Pseudomonas aeruginosa [33]. It was found anxiolytic in rat [34] and contains high amount of

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phenolic compounds, sabinene and terpinen-4-ol [35,36], which have been recognized as

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antioxidant and antibacterial compounds [36–39]. According to the above-mentioned health

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effects of marjoram, it is worthy to assess its potential in fish growth and health boosting.

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Common carp, Cyprinus carpio, is one of the key aquaculture species in the world, with annual

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production of more than 4.1 million tons in 2017. There are reports about A. hydrophila outbreak

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in carp ponds [4], which rise a need for research on this topic. Accordingly, and considering the

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importance of this species, the goal of this research was investigating the growth-promotion,

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antioxidant, immunostimulant effects of oregano in common carp and their association to fish

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resistance against A. hydrophila infection.

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2. Material and methods

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2.1. Diet preparation and feeding trial

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Marjoram leaves were purchased from a local shop and washed with deionized water. After

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drying against a fan blow, the leaves were powdered and mixed with 80% ethanol at a portion of

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1:3 (Weight/Volume). The mixture was occasionally shaken throughout a three-day period; then

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filtered through a Whatman paper to obtain alcoholic extract. The extract was concentrated in

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rotary and kept at -20 ºC until use [40]. There were four diets in this experiment, containing 0

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(control), 100, 200, and 400 mg kg-1 marjoram extract (Table 1) according to Rashidian et al.

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[41].

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Common carp fingerlings weighing 25.44±0.10 g was procured from a local farm and

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transported to laboratory facilities in Karaj, Iran. Total number of 360 fish were stocked in 12

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tanks (filled with 200 L water) at a density of 30 fish per tank and allowed acclimatizing to the

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laboratory conditions for two weeks and fed with the control diet within this period. Then, the

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tanks divided into four triplicated treatments fed either of the aforementioned diets for 60 days.

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The fish were fed trice a day at a rate of 3% of biomass. The biomass in each tank was weighed

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biweekly to adjust the feed amount. Water flow rate was 0.5 L min-1 and the tanks were

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continuously aerated throughout the experiment. Physicochemical parameters of the rearing

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water including temperature, dissolved oxygen, pH, total hardness and total ammonia nitrogen

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were daily measured, being 22.3±1.13 °C, 6.28±0.68 mg L-1, and 7.49±0.73, 184.44±15.63 mg

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L-1 and 0.03±0.006 mg L-1, respectively.

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2.2. Growth performance calculation

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At the end of feeding trial, the fish were bulk-weighed, and growth parameters were measured

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according to the following equations:

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Weight gain (WG; g) = final weight – initial weight

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Specific growth rate (SGR; % d-1) = 100×

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Feed conversion ratio (FCR) =

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2.3.1. Sampling

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Once the 8-week feeding trial finished, feeding was ceased for 24 h and six fish were sampled

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from each treatment. Upon removing the fish from the tanks, they were anesthetized with

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eugenol (100 mg L-1) [19]. Blood samples were withdrawn using 2-mL heparinized syringes and

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collected in plastic tubes. Plasma was separated at 3500 rpm for 10 min centrifugation and kept

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at -80 oC for further analyses.

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Further six fish were caught per treatment for mucus collection. Mucus samples were collected

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as suggested by Hoseinifar et al. [42] through placing fish into polyethylene bags containing 10

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mL of 50 mM NaCl. The bags were gently shaken by hand for approximately 1–2 min. Then, the

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fish were removed out the bag and the residuals were collected in plastic tubes. The residuals

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were centrifuged at 1500 g (4 °C) for 10 min, and the supernatant was stored at -80 °C for

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mucosal analyses.

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2.3.2. Hematological parameters

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Hematological parameters were monitored in the fish whole blood, according to Mazandarani

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and Hoseini [43]. Red blood cells (RBCs) and white blood cells (WBCs) were counted on

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hemocytometer slide. Micro hematocrit method was used to determine hematocrit (Hct) and

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reported as percentage packed cell volume. Hemoglobin (Hb) content was measured using a

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commercial kit (Zistchem, Tehran, Iran) based on cyanomethemoglobin method. The average red

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blood cell volume (MCV), the mean red blood cell hemoglobin (MCH) and the mean blood

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concentration of hemoglobin in the red blood cells (MCHC) parameters were measured as

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follows:

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MCHC= Hb × 10/Hct

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MCV= Hct × 10/RBC (million)

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MCH= Hb × 10/RBC (million)

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2.3.3. Plasma antioxidant parameters

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Plasma superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) were

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determined using Zellbio commercial kits (Zellbio, Veltinerweg, Germany) according to a

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previous study [44]. Plasma SOD was determined based on conversion of superoxide anion to

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hydrogen peroxide method. Plasma CAT was determined based on decomposition rate of

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hydrogen peroxide. Plasma MDA levels were determined based on reaction with thiobarbituric

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acid at 95ºC.

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2.4. Plasma and mucosal immunological parameters

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Plasma and mucosal lysozyme activity were determined according to Taheri Mirghaed et al. [45]

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using hen’s egg white lysozyme (Sigma, USA) for calibration and 0.05 M sodium phosphate

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buffered saline (pH 6.2) as the reaction medium. Briefly, 50 μL of plasma or mucus samples

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were added to 2 mL of a suspension of Micrococcus luteus, (0.2 mg mL-1) in the buffer. Decline

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in the mixture absorbance was read at 450 nm after 0.25 min and 5 min. Microprotein assay

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method was used for quantifying plasma and mucus total immunoglobulin (Ig) level (C-690;

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Sigma). 12% polyethylene glycol solution applied for Ig precipitation and total Ig level presented

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after subtracting protein content before and after precipitation [46]. Alternative complement

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activity (ACH50) was determined based on the hemolysis of sheep red blood cells (SRBC)

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according to Ortuno et al. [47] protocol. The volume of plasma or mucus yielding 50%

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hemolysis was determined and used to calculate the complement activity of the samples (value of

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ACH50 is in units per mL). Mucosal protease activity was measured by the azocasein hydrolysis

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method described by Ross et al. [48]. The activity oof mucosal alkaline phosphatase (ALP) was

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measured using a commercial kit (Pars Azmun Co., Tehran, Iran) [40].

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2.5. Bacterial challenge

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A. hydrophila (AH04) was obtained from Department of Aquatic Animal Health, Faculty of

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Veterinary Medicine, University of Tehran, Tehran, Iran. The bacterium was cultured on

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tryptone soya agar (TSA) medium and harvested in NaCl 0.85% solution. 30 fish from each

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treatment were anesthetized and intraperitoneally injected by 0.1 mL of the bacterium suspension

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at 108 CFU per fish and survival rates were recorded within 10 days [49].

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Dataset was checked for normality and homogeneity of variance by Shapiro-Wilk and Levene

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tests, respectively. The data were analyzed using SPSS software version no. 24.00 (SPSS Inc.,

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Chicago, IL, USA) and represent the mean ± SE (standard error). Differences among the

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treatments were determined by one-way analysis of variance (ANOVA) followed by Duncan's

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multiple range test considering P < 0.05 as significance level.

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3. Results

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According to the results (Table 2), there were significant effects of dietary marjoram extract on

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growth performance of the fish. Dietary supplementation with 200 mg kg-1 marjoram extract

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resulted in significantly higher final weight and weight gain, compared to the other treatments.

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FCR in the fish fed the diets supplemented with 100-400 mg kg-1 marjoram extract was

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significantly lower than the control treatment; the highest value was observed in the fish fed 200

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mg kg-1 marjoram extract. The fish fed 100 and 200 mg kg-1 marjoram extract showed

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significantly higher SGR compared to the control treatment. There was no mortality in any

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treatments throughout the experiment.

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Hematological parameters are presented in Table 3. According to the results, dietary

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supplementation with 200 mg kg-1 marjoram extract significantly increased RBC, in relation to

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the control treatment. All levels of dietary marjoram extract significantly increased blood Hct

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and Hb, compared to the control treatment. Blood MCH and MCV of the fish fed diet containing

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200 and 400 mg kg-1 marjoram extract were significantly higher than the control treatment.

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There were no significant differences in blood MCHC among the treatments.

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Plasma SOD, CAT and MDA levels are presented in Fig. 1. All levels of dietary marjoram

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extract significantly increased plasma SOD activity, in comparison to the control treatment; the

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highest level was related to the fish fed 400 mg kg-1 marjoram extract diet. There was no

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significant difference in plasma CAT activity among the treatments. There was no significant

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difference in plasma MDA levels among the fish fed 100-400 mg kg-1 marjoram extract diets;

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however, these treatments showed significantly lower MDA levels compared to the control

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treatment.

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Humoral immunological parameters are presented in Table 4. Dietary supplementation with 200

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mg kg-1 marjoram extract significantly increased WBC, in relation to the control treatment.

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According to the results, there were no significant differences in plasma total Ig, lysozyme and

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ACH50 between the control and 100 mg kg-1 marjoram extract treatments. However, the fish fed

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diets containing 200 and 400 mg kg-1 marjoram extract exhibited significant increase in these

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parameters, when compared to the control treatment.

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Mucosal immune responses are presented in Fig. 2 and 3. There was no significant difference in

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mucosal protease activity among the treatments. There was no significant difference in mucosal

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ALP and total Ig levels between the control and 100 mg kg-1 marjoram extract treatments. The

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fish fed diets containing 200 and 400 mg kg-1 marjoram extract showed significantly higher

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mucosal ALP and total Ig compared to the control treatment; the highest level was observed in

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the 200 mg kg-1 marjoram extract treatment. There was no significant difference in mucosal

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lysozyme and ACH50 activities among the control, 100 and 400 mg kg-1 marjoram extract

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treatments. The highest mucosal lysozyme and ACH50 activities were observed in the fish fed

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diet containing 200 mg kg-1 marjoram extract.

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Results of challenge with A. hydrophila showed that fish fed 100-400 mg kg-1 marjoram extract

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diets had significantly higher survival than the control treatment; the highest survival was related

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to the 200 mg kg-1 marjoram extract treatment (Fig. 4).

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4. Discussion

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Phytotherapy is a useful means to augment fish growth performance via improvement of gut

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morphology, microbial community, and activity of digestive enzymes [50]. There is no study on

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the effects of dietary marjoram on growth performance of fish; however, other Labiatae plants

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including T. vulgaris and O. vulgare were found to improve fish growth rate and such

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improvements seem to be due to changes in gut morphology [28], microbial community [31,51]

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or increase in digestive enzymes’ activity [31]. Therefore, it may be speculated that the growth-

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promoting effects of dietary marjoram extract were due to its effects on fish gut.

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Hematological parameters are good indicators of fish health and increase in RBC, Hct and Hb

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facilitates tissue oxygenation and elimination of carbon dioxide [52]. Such effects may partly

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have contributed to the better growth performance of fish fed 200 mg kg-1 diet [53]. Such

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relationships between the fish growth performance and hematological parameters have been

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reported in previous studies on other plant extracts [54,55]. Increase in MCV and MCH in the

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fish fed diet containing 200 mg kg-1 marjoram indicates a possible hematopoietic effects of the

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marjoram, as new and young RBC are larger in size and contain higher amount of Hb [56].

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Proper function of antioxidant system is a crucial factor in fish health and plant materials are

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useful additives to augment the system because of occurrence of natural antioxidants. MDA is a

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good indicator of oxidative stress and the present results show that marjoram extract was capable

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with reducing oxidative conditions in the fish. This might be due to the presence of natural

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antioxidants in marjoram such as phenolic compounds, sabinene and terpinen-4-ol [35,36],

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which induce radical scavenging activity [36,39]. Moreover, lower MDA levels might be due to

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higher antioxidant enzymes’ activity (SOD in the present study) that neutralize pro-oxidant

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compounds. The antioxidant effects of the marjoram extract are comparable to those of T.

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vulgaris and O. vulgare, which induced antioxidant enzymes and suppressed oxidative stress

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upon dietary administration [22,23,25,27].

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Leukocytes are important components of fish immune system, which have various

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immunological functions such as phagocytosis and antibody production [43]. Increase in blood

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leukocyte number have been reported upon administration of herbal materials and such increases

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were accompanied by elevated disease resistance [15,57]. Ig are secreted by lymphocytes and/or

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epithelial cells that recognize antigens such as bacteria and higher basal Ig levels has been found

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be associated to higher disease resistance in fish fed diets supplemented with herbal additives

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[58,59]. Blood and mucosal lysozyme are important bactericidal agents and previous studies

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have shown that herbal additives increase the enzymes activity, which was parallel to increase in

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fish resistance to bacterial challenge [58,59]. Complement proteins are produced by fish liver

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and attacks membrane of foreign cells; it has been reported that herbal additives are capable to

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increase fish complement activity, by which, the fish showed higher resistance against bacterial

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challenge [60,61]. Mucosal ALP has been known to be associated to fish immunity due to its

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hydrolytic activity and studies have shown that dietary herbal additives increase mucosal ALP

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activity along with higher disease resistance in fish [58]. There are no studies on the effects of

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marjoram extract on fish humoral and mucosal immune responses and disease resistance.

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However, other plants of Labiatae family showed results comparable to the present study. For

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example, dietary administration T. vulgaris essential oil up-regulated complement and lysozyme

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gene expression and increase survival of fish against A. hydrophila [26]. Moreover, dietary O.

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vulgare and Origanum heracleoticum administration increased lysozyme activity and fish

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resistance against A. hydrophila [22,27].

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Moreover, higher fish survival against the bacterial challenge in the present study might be due

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to improved antioxidant power of the fish, as bacterial infection induces strong oxidative stress

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[62,63]. line with this hypothesis, Abdel-Latif et al. [27] and Zheng et al. [22] reported that

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higher resistance against A. hydrophila in fish fed O. vulgare- and O. heracleoticum-

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supplemented diets was in consort with increased antioxidant power.

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5. Conclusion

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In conclusion, dietary marjoram extract seems a suitable feed additive in common carp that

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improve the fish growth, antioxidant and immune power, and resistance against Aeromonas

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septicemia. According to the results, 200 mg marjoram extract kg-1 is suitable for common carp

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feed formulation.

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Conflicts of interest

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Authors have no conflict of interest to declare for the publication of the present work.

268 Acknowledgment

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This paper has been supported by the RUDN University Strategic Academic Leadership

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Program. This research work was partially supported by Chiang Mai University.

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504

2016-0024.

505

23

506

Table 1. Feedstuffs composition and chemical characteristics of the control diet (Yousefi et al.,

507

2020) Amount (%)

Fishmeal

10

Soybean meal

23

Meat meal

21

Wheat meal

40.8 1

Soybean oil

1

ro

Fish oil

0.7

-p

Lysine Methionine

0.5

re

Vitamin mix

lP

Mineral mix LE

na

Dry matter (%)

1

1 0

90.6

Crude protein (%)

38.9

Crude fat (%)

8.65

ur Jo

508

of

Feedstuffs

Crude ash (%)

6.11

Energy (mj kg-1)

15.6

509

Table 2. Growth performance of common carp fed the experimental diets for 60 days. Values are

510

presented as the mean ± SE. Marjoram extract concentration (mg kg-1) Parameters

Control

100

200

400

Initial weight (g)

25.56±0.11

25.37±0.30

25.31±0.19

25.51±0.23

Final weight (g)

61.56±1.77b

67.36±1.73b

73.56±1.89a

66.41±1.96b

Weight gain (g)

35.99±1.69b

41.99±1.90b

48.25±1.70a

40.90±2.05b

FCR

1.69±0.02a

1.40±0.03b

1.32±0.01c

1.42±0.02b

24

SGR (%/d-1)

1.46±0.04c

1.62±0.05ab

1.77±0.03a

1.59±0.05bc

SR (%)

100±0.00

100±0.00

100±0.00

100±0.00

511

Different letters (a–c) within a row indicate significant differences (P < 0.05).

512 513

Table 3. Hematology indices of common carp fed four experimental diets for 60 days. Values

514

are presented as the mean ± SE.

RBC (× 106 µL-1)

1.37±0.02b

1.39±0.01ab

Hct (%)

23.33±0.88b

26.36±0.85a

28.50±0.76a

28.33±0.88a

Hb (g dL-1)

6.24±0.33b

7.10±0.17a

7.76±0.20a

7.60±0.21a

MCHC (g dL-1)

26.95±2.42

re

200

400

1.45±0.02a

1.43±0.03ab

26.94±0.26

27.32±1.43

26.90±1.39

MCH (pg)

45.43±2.79b

50.88±1.86ab

53.54±0.55a

53.01±1.01a

MCV (fL)

169.45±5.27b

188.98±8.38ab

196.80±8.24a

197.92±8.90a

-p

ro

100

na

Different letters (a–c) in the same row indicate significant differences (P < 0.05).

ur

515

Control

lP

Parameters

of

Marjoram extract concentration (mg kg-1)

Jo

516 517

Table 4. Immunological parameters of common carp fed four experimental diets for 60 days.

518

Values are presented as the mean ± SE. Marjoram extract concentration (mg kg-1) Parameters

519

Control

100

200

400

WBC (× 103 µL-1)

3.97±0.10b

4.11±0.07ab

4.52±0.19a

4.12±0.08ab

Total Ig (mg mL-1)

35.29±1.30c

37.38±1.43bc

41.63±0.83a

40.67±0.74ab

Lysozyme (U mL-1)

27.96±1.45b

31.67±1.30ab

35.39±1.62a

34.70±1.86a

ACH50 (U mL-1)

54.06±1.83b

56.00±1.64ab

60.64±1.99a

61.07±1.28a

Different letters (a–c) in the same row indicate significant differences (P < 0.05).

25

520 45 a

SOD (U/ml)

40 b

35 30

ab

c

25 20 15

of

10 0 100 mg/kg

200 mg/kg

400 mg/kg

-p

Control

ro

5

Treatments

re

521

lP

80

50 40 30

Jo

20

na

60

ur

CAT (U/ml)

70

10

0 Control

522

100 mg/kg

200 mg/kg

Treatments

26

400 mg/kg

18 MDA (nmol/ml)

16

a b

14

b

b

12 10 8 6 4 0 Control

100 mg/kg

of

2 200 mg/kg

ro

Treatments

400 mg/kg

-p

523

Figure 1. The effects of dietary marjoram extract on plasma SOD and CAT activities and MDA

525

level in common carp after 60 days. Bars assigned with different superscripts are significantly

526

different (P < 0.05); Values are presented as the mean ± SE.

529 530

lP

na ur

528

Jo

527

re

524

27

30

Protease (U/ml)

25 20 15 10 5

100 mg/kg

200 mg/kg

400 mg/kg

ro

Control

of

0 Treatments

-p

531

re

16 14

6

ur

4

ab

lP

c

10 8

bc

na

ALP (U/l)

12

a

Jo

2 0

Control

532

100 mg/kg

200 mg/kg

400 mg/kg

Treatments

533

Figure 2. The effects of dietary marjoram extract on skin mucus protease and ALP activities in

534

common carp after 60 days. Bars assigned with different superscripts are significantly different

535

(P < 0.05); Values are presented as the mean ± SE.

536 537

28

14

a

ab

Total Ig (mg/ml)

12 bc

10 8

c

6 4 2 100 mg/kg

200 mg/kg

400 mg/kg

ro

Control

of

0 Treatments

-p

538

re

25

5

ab

ur

10

b

na

15

Jo

Lysozym (U/ml)

b

lP

20

a

0

Control

539

100 mg/kg

200 mg/kg

Treatments

29

400 mg/kg

90

a

b

ab

Control

100 mg/kg

80

ab

ACH50 (U/ml)

70 60 50 40 30 20 0

of

10 200 mg/kg

ro

Treatments

400 mg/kg

-p

540

Figure 3. The effects of dietary marjoram extract on skin mucus total immunoglobulin level,

542

lysozyme activity and alternative hemolytic complement activity (ACH50) in common carp after

543

60 days. Bars assigned with different superscripts are significantly different (P < 0.05); Values

544

are presented as the mean ± SE.

na

lP

re

541

ur

545

Jo

546

Control

100 mg/kg

200 mg/kg

400 mg/kg

SURVIVAL RATE (%)

120 100 80 60 40 20 0 1

2

3

4

5

6

7

8

DAYS POST CHALLENGE

547 30

9

10

548

Figure 4. Percent survival rate of common carp fed different levels of dietary marjoram during a

549

10-day period of exposure to Aeromonas hydrophilla.

Jo

ur

na

lP

re

-p

ro

of

550

31

Highlights Marjoram is a beneficial feed supplement to enhance growth performance

•

Marjoram significantly increase skin mucosal immunity in common carp

•

Marjoram is a beneficial dietary supplement which can improve both hematological and innate immune responses

•

Marjoram could significantly improve survival rate and resistance of common carp against A. hydrophila

Jo

ur

na

lP

re

-p

ro

of

•